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Protein Technology (Defining expressed region (Preserve secondary…

- - - - lipids
      - electroporation
      - nanoparticles
  - - - The fragment and the target DNA must share identical DNA segments at positions intended for Homologous Recombination
      - Recombination is catalyzed by RecA
      - No need for restriction enzymes or ligase
      - Workflow
- - - - E. Coli
        
        Most utilized
        
        Easy to handle, and cheap
        
        Not particularly sophisticated
        
        50% BACTERIA, 10% eukaryotic are expressed
        
        the larger the protein the harder to produce
        
        plasmid vectors
        
        Challenges with protein folding
        
        parameters
        
        Temperature
        
        Oxygen levels
        
        Inclusion bodies
      - B subtilis
        
        Free of endotoxins, Gram+
        
        Secretes proteins to medium
        
        Supports formation of disulfides
        
        No specific requirements for codon use
        
        CHALLENGES
        
        Unstable plasmids
        
        Proteases cause degradation
        
        Less expression plasmids
        
        Poor folding of recombinant proteins
    - - K. pastoris
        
        Metylotrophic yeasts
        
        Simple medium growth conditions
        
        Eukaryotic
        
        Low endogenous protein levels
        
        Challenges
        
        Proteolytic degradation
        
        Clone-to-clone variation
        
        Less tools
        
        Risk of contamination
    - - Baculovirus expression system
        
        Baculovirus used as a vector
        
        Non-essential gene replaced by target protein gene
        
        Recombinant baculovirus genome transfected into incect cell
        
        Infects insects worms in the nauture
        
        Protocol
        
        one setback is the Lytic cyckle
        
        see the protocol in slides
        
        Challenges
        
        Glycosylation and phosphorylation differs
        
        Medium expencive
        
        Contaminations
        
        Fragile cells
    - - CHO
        
        Requires glycine, hypoxanthanine and thymidine
        
        Plasmid transfection
        
        gene of interest + DHFR-gene
        
        DHFR(-) - Missing dihydrofolate reductase
        
        SUMMARY
        
        Excellent for therapeutic proteins
        
        Requires genomic integration and screening
        
        Tedious to set up
        
        Expencive vs E. coli
        
        Post-translational modifications & Efficient folding
        
        Commercially available
  - - - size
      - complexity
      - folding
- - - - Solubility
      - Changes in protein stability/residence time
    - - Specific binding to another biomolecule